Lyophilizable and enhanced compacted nucleic acids

ABSTRACT

Counterions of polycations used to compact nucleic acids profoundly affect shape and stability of particles formed. Shape is associated with differential serum nuclease resistance and colloidal stability. A surrogate for determining such properties that is easy to measure is the turbidity parameter. Shape also affects the suitability and efficacy of compacted nucleic acid complexes for transfecting cells by various routes into a mammalian body. Moreover, counterions such as acetate can protect compacted nucleic acid complexes from adverse effects of lyophilization

[0001] This application claims the benefit of application Ser. Nos.60/287,419 filed May 1, 2001 and 60/207,949 filed May 31, 2000, thedisclosures of which are expressly incorporated herein.

BACKGROUND OF THE INVENTION

[0002] Despite the promise of preclinical models for systemic genetherapy to liver, lung, and other tissues, there is currently nocommercial gene therapy product on the market. The failure of most humangene therapy clinical trials to treat metabolic disorders and cancer hasbeen ascribed to the relative inefficiency of viral and non-viral genetransfer systems. Viral vectors have been used for most gene therapystudies because of their ability to efficiently infect cells in tissueculture. However, an enormous payload of particles needs to be appliedin an intravenous injection to transduce cells in vivo, and toxicitiesof viral vectors are well documented [1], including a recent lethaltoxicity that occurred following a portal vein injection of recombinantadenovirus [2]. In contrast, non-viral systems are generally felt to besafe although inefficient. There is a growing consensus that non-viralsystems will be the vector of choice for in vivo applications once genetransfer efficiency is improved.

[0003] Several barriers restrict non-viral methods of gene transfer,including: i) particle stability in blood and interstitial tissues; ii)ability of the gene transfer particle to exit capillaries and travel toparenchymal cells; iii) cell entry via receptor-mediated endocytosis orcell fusion; iv) stability in and escape from endosomal and lysosomalcompartments; v) diffusion rate in the cytoplasm; vi) nuclear poretransit; and vii) “uncoating” of DNA to permit biological function inthe nucleus. For example, numerous publications have documented thefailure of non-viral methods to transfect post-mitotic, growth-arrestedcells [3-11], presumably because the intact nuclear membrane ofnon-dividing cells restricts entry of naked DNA into the nucleus via the25 nm nuclear pore [12-13].

[0004] Thus there is a continuing need in the art for improvedformulations and methods for delivery of genes to animals and humans. Inaddition, there is a need in the art for formulations which will bestable to storage and retain biological activity.

SUMMARY OF THE INVENTION

[0005] These and other objects of the invention are provided by one ormore of the embodiments disclosed below. In one embodiment of theinvention a method of estimating the colloidal stability of apreparation of compacted nucleic acids is provided. A turbidityparameter of a solution of compacted nucleic acid is determined. Theturbidity parameter is defined as the slope of a straight line obtainedby plotting log of apparent absorbance of light versus log of incidentwavelength of the light. The wavelength used is between about 330 nm and420 nm. A preparation is identified as colloidally stable if a turbidityparameter of less than −3 is determined. A preparation is identified ascolloidally unstable if a turbidity parameter of greater than or equalto −3 is determined.

[0006] According to another aspect of the invention a non-naturallyoccurring composition comprising unaggregated nucleic acid complexes isprovided. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. The polycation moleculeshave a counterion selected from the group consisting of acetate,bicarbonate, and chloride. The complex is compacted to a diameter whichis less than (a) double the theoretical diameter of a complex of saidsingle nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere, or (b) 30 nm, whichever is larger. Optionally, the oneor more polycation molecules of the unaggregated nucleic acid complexesare CK15-60P10, wherein acetate is used as a counterion. CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, with a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal attached to the cysteine residue.

[0007] According to another aspect of the invention a method ofpreparing a composition comprising unaggregated nucleic acid complexesis provided. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. The polycation moleculeshave a counterion selected from the group consisting of acetate,bicarbonate, and chloride. The complex is compacted to a diameter whichis less than (a) double the theoretical diameter of a complex of saidsingle nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere, or (b) 30 nm, whichever is larger. The nucleic acid ismixed with the polycation having acetate, bicarbonate, or chloride as acounterion, at a salt concentration sufficient for compaction of thecomplex. Optionally, the one or more polycation molecules of theunaggregated nucleic acid complexes are CK15-60P10, wherein acetate isused as a counterion. CK15-60P10 is a polyamino acid polymer of oneN-terminal cysteine and 15-60 lysine residues, with a molecule ofpolyethylene glycol having an average molecular weight of 10 kdalattached to the cysteine residue.

[0008] An additional embodiment of the invention is provided as a methodof preparing a composition comprising unaggregated nucleic acidcomplexes. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. A nucleic acid moleculeis mixed with a polycation molecule at a salt concentration sufficientfor compaction of the complex to a diameter which is less than doublethe theoretical minimum diameter of a complex of said single nucleicacid molecule and a sufficient number of polycation molecules to providea charge ratio of about 1:1, in the form of a condensed sphere, or 30nm, whichever is larger. Unaggregated nucleic acid complexes are formed.Optionally, the one or more polycation molecules of the unaggregatednucleic acid complexes are CK15-60P10, wherein acetate is used as acounterion. CK15-60P10 is a polyamino acid polymer of one N-terminalcysteine and 15-60 lysine residues, with a molecule of polyethyleneglycol having an average molecular weight of 10 kdal attached to thecysteine residue.

[0009] Also provided by the present invention is a non-naturallyoccurring composition comprising unaggregated nucleic acid complexes.Each complex consists essentially of a single nucleic acid molecule andone or more polycation molecules. The polycation molecules have acounterion selected from the group consisting of acetate, bicarbonate,and chloride. The nucleic acid molecule encodes at least one functionalprotein. Said complex is compacted to a diameter which is less thandouble the theoretical minimum diameter of a complex of said singlenucleic acid molecule and a sufficient number of polycation molecules toprovide a charge ratio of about 1:1, in the form of a condensed sphere,or 30 nm, whichever is larger. Optionally, the one or more polycationmolecules of the unaggregated nucleic acid complexes are CK15-60P10,wherein acetate is used as the counterion. CK15-60P10 is a polyaminoacid polymer of one N-terminal cysteine and 15-60 lysine residues, witha molecule of polyethylene glycol having an average molecular weight of10 kdal attached to the cysteine residue.

[0010] Another non-naturally occurring composition comprisingunaggregated nucleic acid complexes is also provided. Each complexconsists essentially of a single double-stranded cDNA molecule and oneor more polycation molecules. Said polycation molecules have acounterion selected from the group consisting of acetate, bicarbonate,and chloride. The cDNA molecule encodes at least one functional protein.The complex is compacted to a diameter which is less than double thetheoretical minimum diameter of a complex of said single cDNA moleculeand a sufficient number of polycation molecules to provide a chargeratio of about 1:1, in the form of a condensed sphere, or 30 nm,whichever is larger. The nucleic acid complexes are optionallyassociated with a lipid. Optionally, the one or more polycationmolecules of the unaggregated nucleic acid complexes are CK15-60P10,wherein acetate is used as the counterion. CK15-60P10 is a polyaminoacid polymer of one N-terminal cysteine and 15-60 lysine residues, witha molecule of polyethylene glycol having an average molecular weight of10 kdal attached to the cysteine residue.

[0011] Another non-naturally occurring composition comprisingunaggregated nucleic acid complexes is provided by the presentinvention. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. The polycation moleculeshave a counterion selected from the group consisting of acetate,bicarbonate, and chloride. The nucleic acid molecule encodes at leastone antisense nucleic acid. The complex is compacted to a diameter whichis less than double the theoretical minimum diameter of a complex ofsaid single nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere, or 30 nm, whichever is larger. Optionally, the one ormore polycation molecules of the unaggregated nucleic acid complexes areCK15-60P10, wherein acetate is used as the counterion. CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, with a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal attached to the cysteine residue.

[0012] According to another aspect of the invention a non-naturallyoccurring composition comprising unaggregated nucleic acid complexes isprovided. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. The polycation moleculehas a counterion selected from the group consisting of acetate,bicarbonate, and chloride. The nucleic acid molecule is an RNA molecule.The complex is compacted to a diameter which is less than double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere, or 30 nm,whichever is larger. Optionally, the one or more polycation molecules ofthe unaggregated nucleic acid complexes are CK15-60P10, wherein acetateis used as the counterion. CK15-60P10 is a polyamino acid polymer of oneN-terminal cysteine and 15-60 lysine residues with a molecule ofpolyethylene glycol having an average molecular weight of 10 kdal isattached to the cysteine residue.

[0013] Another aspect of the invention provided here is a method ofpreparing a composition comprising unaggregated nucleic acid complexes.Each complex consists essentially of a single nucleic acid molecule andone or more polycation molecules. A nucleic acid molecule is mixed witha polycation molecule in a solvent to form a complex. The mixing isperformed in the absence of added salt, whereby the nucleic acid formssoluble complexes with the polycation molecule without formingaggregates. Each complex consists essentially of a single nucleic acidmolecule and one or more polycation molecules. The complexes have adiameter which is less than double the theoretical minimum diameter of acomplex of the single nucleic acid molecule and a sufficient number ofpolycation molecules to provide a charge ratio of about 1:1, in the formof a condensed sphere, or 30 nm, whichever is larger. The polycation hasacetate, bicarbonate, or chloride as a counterion. Optionally, the oneor more polycation molecules of the unaggregated nucleic acid complexesare CK15-60P10, wherein acetate is used as the counterion. CK15-60P10 isa polyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues with a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal is attached to the cysteine residue.

[0014] Finally, the present invention provides a method of preventing ortreating a disease or other clinical condition in a subject. Aprophylactically or therapeutically effective amount of a composition isadministered intramuscularly or to the lung. The composition comprises:unaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more polycation molecules,said polycation molecule having acetate, chloride, or bicarbonate as acounterion, wherein said complex is compacted to a diameter which isless than (a) double the theoretical minimum diameter of a complex ofsaid single nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere, or (b) 30 nm, whichever is larger. The nucleic acid isone whose integration, hybridization or expression within target cellsof the subject prevents or treats the disease or other clinicalcondition. Optionally, the one or more polycation molecules of theunaggregated nucleic acid complexes are CK15-60P10, wherein acetate isused as the counterion. CK15-60P10 is a polyamino acid polymer of oneN-terminal cysteine and 15-60 lysine residues with a molecule ofpolyethylene glycol having an average molecular weight of 10 kdal isattached to the cysteine residue.

[0015] The present invention thus provides the art with improvedanalytical and therapeutic techniques for delivery of DNA to cells byproviding compacted nucleic acid compositions having improved stabilityand transfectability properties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows intramuscular (IM) injection results using TFA(trifluoroacetate) and acetate as counterions for polylysine used tocompact DNA.

[0017]FIG. 2 shows intramuscular injection results using TFA(trifluoroacetate) as counterion for polylysine used to compact DNA.

[0018]FIG. 3 shows intramuscular injection results using acetate ascounterions for polylysine used to compact DNA.

[0019]FIG. 4 shows intramuscular injection results using acetate ascounterions for polylysine used to compact DNA.

[0020]FIG. 5 shows a variety of parameters varying and theireffectiveness in IM injections, including size of polylysine (CK),polyethylene glycol substitution.

[0021]FIG. 6 shows intra-tracheal instillation of 100 ug naked and 100ug compacted DNA compared as to amount of expression in the lung of theinstilled gene (luciferase) as a function of time after gene transfer.

[0022]FIG. 7A shows intra-tracheal instillation of naked and compactedDNA compared as to amount of expression in the lung of the instilledgene (luciferase) as a function of time after gene transfer. FIG. 7Bshows plot of data above background from FIG. 7A.

[0023]FIG. 8 shows turbidity parameter plots as a function of size ofpolylysine used in compaction and counterion

[0024]FIG. 9A, FIG. 9B, and FIG. 9C show a comparison of serumstability, turbidity parameter, and sedimentation, for variousformulations of compacted nucleic acids. FIG. 9D tabulates the results.

[0025]FIG. 10 shows the influence of counterion on the morphology ofPEG-substituted CK30 compacted DNA as shown under the electronmicroscope.

[0026]FIG. 11 shows the stability of PLASmin™ DNA upon freezing andlyophilization. Particles were tested with sucrose, trehalose, or noexcipient. Particles were tested with and without polyethylene glycol,and with TFA or acetate as the counterion. DNA stability was assessed bya low (3400×g×1 min) spin to pellet aggregates, and monitoring theabsorbance of DNA in the supernatant. Stability with acetate as thecounterion surpassed other formulations in the absence of excipient.

[0027]FIG. 12 shows assessment of the turbidity parameter before andafter lyophilization using various excipients, counterions, and with orwithout polyethylene glycol. Sucrose and trehalose are very effective inmaintaining the properties of the pre-lyophilization particles.PEG-acetate similarly was effective in maintaining the properties.

[0028]FIG. 13 shows a visualization of particles under the electronmicroscope. For particles made with CK30-PEG10k acetate in the presenceof 0.5 M trehalose, the rod-like compacted particles look identicalbefore and after lyophilization and rehydration.

[0029]FIG. 14 shows a visualization of particles under the electronmicroscope. For particles made with CK30 TFA in the presence of 0.5Msucrose, the ellipsoidal particles of compacted DNA look identicalbefore and after lyophilization and rehydration.

[0030]FIG. 15 shows the results of gene transfer experiments usinglyophilized PLASmin™ complexes. Luciferase enzyme was encoded by thecomplexes and its activity was measured as a means of monitoring genetransfer. While sucrose and trehalose were effective in protecting thegene transfer activity to all particles, particles which containedpolyethylene glycol (10 kdal) and acetate as a counterion weresurprisingly stable to lyophilization, even in the absence ofcryoprotectant excipient (disaccharide).

[0031]FIG. 16 shows a comparison of the colloidal stability of CK30P10Kand CK45P10K DNA complexes compacted using various counterions in 0.9%NaCl. Colloidal stability is evaluated by sedimentation and turbidityparameter.

[0032]FIG. 17 shows an electron micrograph of plasmid DNA compacted byCK45P10with chloride as a counterion. Magnification 40,000. The barshows 100 nm.

[0033]FIG. 18 shows an agarose gel electrophoresis of DNA compacted byPEG-ylated polylysine (CK30P10K) with various counterions. The influenceof counterions on the effective net charge of the condensed DNA can beseen by the migration of the compacted DNA through the gel. FIG. 18 alsoshows the serum stability of the CK30P10K-DNA complexes with each of thedifferent counterions.

[0034]FIG. 19 shows in vivo expression of luciferase plasmid compactedby various counterion forms of PEG-ylated polylysine (CK30P10K) afterintramuscular application. Each point represents one animal. The solidline indicates background signal of luciferase assay. Dose was 100 μgDNA.

[0035]FIG. 20 shows in vivo expression of luciferase plasmid compactedby various forms of PEG-ylated polylysine after intranasal application.Acetate, bicarbonate, and TFA forms of CD30P10K and chloride form ofCK45P10K were used. The acetate formulation was prepared either insaline or water. Each point represents one animal. The solid lineindicates background signal of luciferase assay. Dose was 100 μg DNA.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The disclosures of U.S. Pat. Nos. 5,844,107, 5,877,302,6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900 andapplications Ser. Nos. 60/145,970, 09/722,340, 09/311,553 and 60/207,949are expressly incorporated herein.

[0037] Counterions of polycations used to compact nucleic acidsprofoundly affect shape of particles formed. Shape is associated withdifferential serum nuclease resistance and colloidal stability. Asurrogate for determining such properties which is easy to measure isthe turbidity parameter. Moreover, shape affects the suitability andefficacy of compacted nucleic acid complexes for transfecting cells byvarious routes into a mammalian body.

[0038] The counterion used in making compacted nucleic acid complexesalso has a significant effect on the stability of the complexes tolyophilization. Since lyophilization is a common process to renderbiologicals readily transportable and storage stable, this finding hassignificant ramifications. Typically, polyamino acid polymers containtrifluoroaceate (TFA) as a counterion. However, this counterion is farless beneficial than acetate for purposes of lyophilization of nucleicacid polymers, as shown below. Particles made using acetate retain theirunaggregated nature, i.e., stay in solution better, after lyophilizationand rehydration, retain their shape, and retain their gene transferpotential.

[0039] Particles according to the present invention contain nucleicacids, preferably a single nucleic acid molecule. The nucleic acid maybe DNA or RNA, may be double or single-stranded, may be protein codingor anti-sense coding or non-coding. Nucleic acids also include analogsof RNA and DNA which are modified to enhance the resistance todegradation in vivo. A preferred analogue is a methylphosphonateanalogue of the naturally occurring mononucleosides. More generally, themononucleoside analogue is any analogue whose use results inoligonucleotides which have the advantages of (a) an improved ability todiffuse through cell membranes and/or (b) resistance to nucleasedigestion within the body of a subject (Miller, P. S. et al.,Biochemistry 20:1874-1880 (1981)). Such nucleoside analogues arewell-known in the art. The nucleic acid molecule may be an analogue ofDNA or RNA. The present invention is not limited to use of anyparticular DNA or RNA analogue, provided it is capable of fulfilling itstherapeutic purpose, has adequate resistance to nucleases, and adequatebioavailability and cell take-up. DNA or RNA may be made more resistantto in vivo degradation by enzymes, e.g., nucleases, by modifyinginternucleoside linkages (e.g., methylphosphonates or phosphorothioates)or by incorporating modified nucleosides (e.g., 2′-0-methylribose or1′-alpha-anomers). The methods used for forming the particles are asdisclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835,5,972,901, 6,200,801, and 5,972,900 and applications Ser. Nos.60/145,970, 09/722,340, 09/311553 and 60/207949.

[0040] Polycations according to the present invention preferablycomprise polyamino acids such as polylysine and derivatives ofpolylysine. The polycation may contain from 15-60 lysine residues,preferably in the ranges of 15-30, 30-45, or 45-60 residues. Preferredderivatives of polylysine are CK15, CK30, CK45, which have an additionalcysteine residue attached to polylysine polymers of length 15, 30, and45 residues, respectively. Other amino acids can be readily attached topolylysine without departing from the spirit of the invention. Otherpolycationic amino acid polymers can be used such as polyarginine, orcopolymers of arginine and lysine. Polymers of non-protein amino acids,such as ornithine or citrulline, could also be used. Anypharmaceutically approved or appropriate polycation can be usedincluding but not limited to protamine, histones, polycationic lipids,putrescine, spermidine, spermine, peptides, and polypeptides. Thepolycation may also contain a targeting moiety, which is typically aligand which binds to a receptor on a particular type of cell. Thetargeting ligand may be a polyamino acid or other chemical moiety.Specificity of interaction of the ligand and the receptor is importantfor purposes of targeting.

[0041] Conditions for making compacted nucleic acid particles aredisclosed in the aforementioned patents and applications. The conditionsmay include from 0-1 M salt. The preferred salt is NaCl. Otherchaotropic salts can be used as long as they are tolerated by the animal(or cells) to which they will be administered. Suitable agents includeSodium sulfate (Na.sub.2 SO.sub.4), Lithium sulfate (Li.sub.2 SO.sub.4),Ammonium sulfate ((NH.sub.4).sub.2 SO.sub.4, Potassium sulfate (K.sub.2SO.sub.4), Magnesium sulfate (MgSO.sub.4), Potassium phosphate (KH.sub.2PO.sub.4), Sodium phosphate (NaH.sub.2 PO.sub.4), Ammonium phosphate(NH.sub.4 H.sub.2 PO.sub.4), Magnesium phosphate (MgHPO.sub.4),Magnesium chloride (Mg Cl.sub.2), Lithium chloride (LiCl), Sodiumchloride (NaCl), Potassium chloride (KCl), Cesium chloride (CaCl),Ammonium acetate, Potassium acetate, Sodium acetate, Sodium fluoride(NaF), Potassium fluoride (KF), Tetramethyl ammonium chloride (TMA-Cl),Tetrabutylammonium chloride (TBA-Cl), Triethylammoniym chloride(TEA-Cl), and Methyltriethylammonium chloride (MTEA-Cl).

[0042] If a Target Cell Binding Moiety (TBM) is used, it must bindspecifically to an accessible structure (the “receptor”) of the intendedtarget cells. It is not necessary that it be absolutely specific forthose cells, however, it must be sufficiently specific for the conjugateto be therapeutically effective. Preferably, its cross-reactivity withother cells is less than 10%, more preferably less than 5%.

[0043] There is no absolute minimum affinity which the TBM must have foran accessible structure of the target cell, however, the higher theaffinity, the better. Preferably, the affinity is at least 10.sup.3liters/mole, more preferably, at least 10.sup.6 liters/mole.

[0044] The TBM may be an antibody (or a specifically binding fragment ofan antibody, such as an Fab, Fab, V.sub.M, V.sub.L or CDR) which bindsspecifically to an epitope on the surface of the target cell. Methodsfor raising antibodies against cells, cell membranes, or isolated cellsurface antigens are known in the art: (a). production of immune spleencells: immunization with soluble antigens Hurrell, J. G. R. (1982)Monoclonal Antibodies: Techniques and Applications. CRC Press, BocaRaton, Fla. (b). immunization with complex antigens: membranes, wholecells and microorganisms. Hurrell, J. G. R. (1982) MonoclonalAntibodies: Techniques and Applications. CRC Press, Boca Raton, Fla.(c). production of monoclonal supernatants and ascites fluids. Andrew,S. M. and Titus, J. A. (1991). Purification of Immunoglobulin G. inCurrent Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H.J. Margulies, E. M. Shevach and W. Strober, ed.) pp. A.3.9-A.3.12.Greene Publishing Wiley-Interscience, New York. (d). production ofpolyclonal antiserum in rabbit. Garvey J. S., Cremer, N. E. andSussdorf, D. H (eds) (1977) Methods in Immunology: A Laboratory Text forInstruction and Research, Third Edition. W. A. Benjamin, North Hampton,Mass. (e). production of anti-peptide antibodies by chemical coupling ofsynthetic peptides to carrier proteins Jemmerson, R., Morrow, P. I.,Klinman, N. I and Patterson, Y. (1985). Analysis of an evolutionaryconserved site on mammalian cytochrome C using synthetic peptides. Proc.Natl Acad. Sci, U.S.A. 82, 1508-1512.

[0045] The TBM may be a lectin, for which there is a cognatecarbohydrate structure on the cell surface. The target binding moietymay be a ligand which is specifically bound by a receptor carried by thetarget cells. One class of ligands of interest are carbohydrates,especially mono- and oligosaccharides. Suitable ligands includegalactose, lactose and mannose. Another class of ligands of interest arepeptides (which here includes proteins), such as insulin, epidermalgrowth factor(s), tumor necrosis factor, prolactin, chorionicgonadotropin, FSH, LH, glucagon, lactoferrin, transferrin,apolipoprotein E, gp120 and albumin. The following table lists preferredtarget binding moieties for various classes of target cells: TargetCells Target Binding Moiety liver cells galactose Kupffer cells mannosemacrophages mannose lung Fab fragment vs. polymeric immunoglobulinreceptor (Pig R) adipose tissue insulin lymphocytes Fab fragment vs. CD4or gp120 enterocyte Vitamin B12 muscle insulin fibroblastsmannose-6-phosphate nerve cells Apolipoprotein E

[0046] Use of a target binding moiety is not strictly necessary in thecase of direct injection of compacted nucleic acid complex. The targetcell in this case is passively accessible to the compacted complex bythe injection of the complex to the vicinity of the target cell. Targetbinding moieties can be attached to lysine residues, cysteine residues,or PEG using covalent or non-covalent interactions.

[0047] It has been found that the counterion provided in associationwith the polycation profoundly affects shape, and that shape isassociated with physiologically important properties for delivery ofnucleic acids. For example, trifluoroacetate (TFA) particles formspheroids and short rods of less than about 50 nm. Acetate leads tolonger rods of 100 to 200 nm. Chloride leads to particles which arelonger and skinnier than acetate particles. Bicarbonate leads to amixture of rods of 100-200 nm and toroids. Any physiologically andpharmacologically acceptable counterion can be used with the polycation.Bromine is typically supplied with reagent grade polylysine. It isbelieved that bromine is inferior to other cations as described herein,especially with respect to physiological acceptability. Counterions canbe supplied to or substituted on polycations by means of chromatographyor dialysis, for example. For example, the polycation can be bound to anion exchange resin and eluted with the desired counterion. Any methodknown in the art can be used for this purposed. Interestingly, it hasbeen found that once a particle has been compacted into a particularshaped particle, removal and replacement of the counterion, such as bydialysis, does not significantly alter the shape once assumed. Thus afavorable shape can be obtained with a particle using a non-optimumcounterion for physiological purposes and the counterion can be replacedwith a superior counterion, while retaining the shape obtained duringcompaction with the original counterion. The favorable affects onnucleic acids of the counterions may not require compaction. Thus thepolycations and counterions can be used with non-compacted nucleic acidsas well.

[0048] The behavior of these different shaped particles in gene deliveryin animals varies significantly. Acetate particles are superior, forexample, to TFA particles for delivery to muscle and lung. Delivery toother locations in the body may also be accomplished. These include,without limitation, administrations which are intratracheal, byinhalation, intradermal, topical, by eyedrops, subcutaneous,intrathecal, by enema, enteral, intravenous, intraarterial,intralymphatic, intraperitoneal, intrapleural, intravesicular,intraarticular, intracardiac, intracranial, intratumor, direct to anorgan, by eardrops, by nosedrops, intraurethral, endoscopically to theupper gastrointestinal tract, to the sigmoid, or to the colon, bycystoscopy, by thorascope, by arthroscope, by mediastinoscopy, byendoscopic retrograde chlolangiopancreatography, by Omaya reservoir, byangiography including cardiac catheterization and cerebral angiography,intrauterine, intravaginal, to the bone marrow, to hair follicles, tothe vitreous and aqueous humor, to the sinuses, to the ureter/pelvis ofthe kidney, to the fallopian tube, and to lymph nodes.

[0049] The complexes have a diameter which is less than double thetheoretical minimum diameter of a complex of the single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere. For thepurposes of this invention, “about 1:1” encompasses from 1.5:1 to 1:1.5.

[0050] Turbidity parameter can be assessed by determining the absorbanceof a composition. In a preferred embodiment a Zeiss MCS501 UV-Visspectrometer is used. Other spectrometers as are known in the art can besubstituted. Suitable wavelengths for collection absorbance measurementsare between about 330 nm and 420 nm.

[0051] The invention is explained in particular applications in theexamples which follow.

EXAMPLES Example 1

[0052] Resistance to serum nucleases is, among other properties, animportant feature of any effective gene therapy vector designed to beadministered systemically. Ideally, engineering this resistance shouldnot compromise other desirable properties of a vector, such as its smallsize and colloidal stability. We have developed reagents and methodsthat permit us to reproducibly compact plasmid DNA withpolylysine-polyethylene glycol (PEG) conjugates to form small particleshaving defined morphology (PLASmin™ complexes). Some of theseformulations are stable in serum and do not aggregate in physiologicsaline. By changing components and conditions of the compactionprocedure, size and shape of the particles can be modified. To evaluatepotential correlations between serum stability and the physical state ofPLASmin™ complexes, we have prepared a matrix of 24 formulations usingpolylysines of various lengths and substituted with PEG to variousextents. FIG. 9D. Polylysines having exactly 15, 30, and 45 residueswere obtained by solid-phase synthesis. These polymers contained anN-terminal cysteine residue that was used to conjugate PEG. Variousmixtures of PEG-substituted and non-substituted polylysines we re usedto obtain different PLASmin™ complexes. Stability of the complexes in75% mouse serum was tested by incubating compacted DNA at 37° C. for upto 5 days and determining half-life of DNA degradation. Simultaneously,physical characteristics of the complexes in 150 mM NaCl weredetermined. Morphology was visualized by transmission electronmicroscopy (FIG. 10 and FIG. 17). DNA condensed with acetate andbicarbonate salts of CK30 polylysine assumed forms of long (100-300 nm)and narrow (10-20 nm) rods and relaxed toroids (˜50-100 nm diameter,10-20 nm width); the TFA salt resulted in much shorter rods (<60 nm by20-30 nm) and small globules (20-30 nm); the chloride form of CK30 didnot compact DNA at all (FIG. 10), while CK45/chloride (FIG. 17) gaveresults similar to CK30/acetate. Colloidal instability (tendency toaggregate) was evaluated by a sedimentation assay. Additionally, lightscattering of solutions containing PLASmin™ complexes was measured andexpressed as a turbidity parameter (FIG. 8). We found that all PLASmin™complexes (FIG. 9A) were much more stable in serum than naked DNA. Thehalf-life for compacted DNA ranged from ˜2-17 hr, while naked DNA wascompletely digested within a few minutes. We also found a correlation(r²=0.77) between half-life of degradation and colloidal instability ofPLASmin™ complexes: particles that tended to aggregate were moreresistant to nucleases. The tendency to aggregate also correlated withmorphology of the complexes: rod-like complexes did not aggregate; thus,they all showed very similar serum stability, independent of theircomposition (t_(½)˜2-5 hr). In contrast, spherical complexes showedvarious extents of tendency to aggregate depending on polylysinechain-length and PEG content. There was little difference in serumstability between small globules and rod-like particles. In agreementwith the prediction that aggregated particles should scatter variouslight wavelengths differently than small complexes, we found a goodcorrelation (r²=0.88) between colloidal instability of PLASmin™complexes and turbidity of their solutions (FIG. 9B): stable complexeshad turbidity parameter around −4 to −5 (in accordance with the Rayleighlaw), while for the largest and least stable particles this valueincreased to −1.3. Consequently, the turbidity parameter also correlatedwith the half-life of DNA degradation in serum (r²=0.73; FIG. 9C). Thus,we conclude that the turbidity parameter, which is easy to determine,can be conveniently used to preliminarily screen various formulations ofcompacted DNA and predict their colloidal stability as well as serumstability.

Example 2

[0053] Effective gene transfer to lung would facilitate therapies forpulmonary diseases, such as cystic fibrosis, and may provide a potentmeans for administering mucosal vaccines. Although direct instillationof naked DNA into mouse airways generates measurable transgeneexpression, the level of expression is low, and the duration ofexpression is short. We have developed reagents and formulation methodsthat compact single molecules of plasmid DNA into 20-25 nm particles(PLASmin™ complexes). Unlike naked DNA, these complexes are protectedfrom nuclease digestion and are stable in serum. Additionally, PLASmin™complexes do not aggregate in physiologic saline and can be concentratedto over 12 mg/ml of DNA. To determine if PLASmin™ complexes wouldgenerate significant levels of gene expression in lung, we instillednaked and PLASmin™ complexes into the lungs of C57BL/6J mice via directintratracheal administration. These compacted particles consisted ofplasmid DNA and PEG-substituted polylysine polymers consisting of 30lysine residues. The plasmid construct encoded a luciferase reportergene transcriptionally controlled by a CMV enhancer, an elongationfactor 1-alpha (EF1-alpha) promoter, EF1-alpha intron 1, the RU5translational enhancer from HTLV I, and an SV40 late polyadenylationsignal. A DNA dose of 100 ug was administered in 25 or 50 ul of 150 mMNaCl. At 2, 4, 5, or 12 days following gene transfer, extracts wereprepared from both lungs and luciferase activity was measured asrelative light units per mg of protein (FIG. 6). Whereas naked DNAgenerated a signal of approximately 4,000 RLU/mg on day 2 and 1,100RLU/mg on day 4, PLASmin™ complexes generated approximately 1,100,000RLU/mg on day 2, and 630,000 rlu/mg on day 4. Gene expression persistedfor at least 12 days after gene transfer, although at lower levels.These compacted DNA particles produced 400-fold enhanced gene expressioncompared to naked DNA on day 2, and over 1,300-fold improved geneexpression on day 4. In contrast to whole lung extracts, less geneexpression was noted in trachea, and no expression in liver (data notshown). In dose response studies, peak levels of transgene expressionwas observed using a 100 ug dose (FIG. 7). In summary, we havedetermined that PLASmin™ complexes effectively deliver and expresstransgenes in mouse lung following direct intra-tracheal administration.In studies in progress, the beta-galactosidase reporter gene is beingutilized to define the cell type(s) being transfected. PLASmin™complexes may provide an appropriate gene transfer method for diversepulmonary diseases and/or mucosal vaccines.

Example 3

[0054] Gene transfer in muscle cells following an intramuscularinjection provides a means of safe and effective vaccination, andprovides therapeutic levels of recombinant proteins, such as factor IX,factor VIII, or alpha-1 anti-trypsin.

[0055] To optimize formulations of PLASmin™ DNA for intramuscularadministration, various preparation of compacted DNA encoding theluciferase reporter gene were administered to CD2 mice by singleinjection in the tibialis anterior muscle. Gene expression was assayedat various days post gene transfer and is presented as relative lightunits (RLU)/mg protein. In FIG. 1, expression of compacted DNAformulated with the acetate salt of CK30 polycation (complexed with PEG10 kD) was enhanced, as measured by luciferase activity on both days 1and 3, compared to other preparations of DNA formulated with the TFAsalt of CK30 or CK45. To define further the roles of counterion type,length of polylysine, and percent substitution of polyethylene glycol(PEG), additional experiments were conducted. Animals received IMinjections of TFA complexes consisting of either CK30 or CK45, and PEGsizes of either 5 or 10 kD. FIG. 2) Luciferase activity wassignificantly less than that observed for CK30, PEG 10 kD, acetatecomplexes in FIG. 1. The enhanced gene expression of complexes preparedusing the acetate salt of CK30, PEG 10 kD, was confirmed. (FIG. 3) Inthis experiment, the CK30 polycation generated better luciferaseactivity than the CK45 polymer, and CK30 yielded higher levels ofluciferase activity when complexed with 10 kD rather than 5 kD PEG. Theduration of gene expression produced by acetate complexes consisting ofeither CK30 or CK45, both complexes with PEG 10 kD, were next evaluated,and the results are shown in FIG. 4. In this study, the CK30 polycationgave the best level of reporter gene activity, and the level of activitywas better on day 7 than days 1 or 3. A variety of acetate complexeswere tested for gene activity as shown in FIG. 5. These formulationsincluded CK15, CK30, and CK45 polycations complexed with variouspercentages of PEG 10 kD. A time course to 30 days was performed.Although gene expression on days 1, 3, and 7 appeared better using CK15compared to CK30, the particle sizes of some CK15 complexes were largerthan 30 nm or two times the theoretical diameter of a complex of saidsingle nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere. For days 1, 3, 7, and 15, at least one preparation ofCK30 compacted DNA was superior to any CK45 preparation. For CK30, the100% PEG 10 kD complexes generated better reporter gene activity thaneither the 70% or 40% substitutions. In summary, the best formulation ofcompacted DNA in these studies was the acetate salt of CK30 polycationhaving a 100% substitution with PEG 10 kD.

Example 4

[0056] Prior to injection, animals are anesthetized by intraperitonealinjection with a rodent cocktail of ketamine, xylazine, andacepromazine. A volume of 150 ul anesthetic is administered per mouse,at a concentration of 21.5 mg/ml ketamine, 10.7 mg/ml xylazine, and 0.36mg/ml acepromazine. The final dose is 0.32 mg ketamine, 1.6 mg xylazine,and 0.054 mg acepromazaine per mouse.

[0057] A volume of 25 ml of each plasmid DNA formulation is administeredintratracheally to each animal using a 22-gauge needle. A plasticcatheter is placed in the trachea of the mice via a percutaneousapproach. The resulting does per animal is 300 ug, 100 ug, 30 ug, and 10ug DNA per mouse.

[0058] After injection, animals are anesthetized by carbon dioxide andsacrificed. The animals are bled and rinsed intra-arterially withphosphate buffered saline. The lungs, trachea, and liver are isolatedand rinsed in the saline. Tissue samples are immediately frozen onliquid nitrogen, and then stored at −70° C.

[0059] Lung tissue is homogenized using Polytron in lysis buffer.Protein concentration is determined. Luciferase activity of thehomogenates is determined by luciferase assay.

Example 5

[0060] The stability of PLASmin™ DNA upon freezing and lyophilizationwas assessed. Particles were tested with sucrose, trehalose, or noexcipient. Particles were tested with and without polyethylene glycol,and with TFA or acetate as the counterion to the polyethylene glycol.DNA stability was assessed by a low (3400×g×1 min) spin to pelletaggregates, and monitoring the absorbance of DNA in the supernatant. SeeFIG. 11. Stability of the complexes with acetate as the counterionsurpassed other formulations in the absence of excipient.

Example 6

[0061] The turbidity parameter is defined as the slope of a straightline obtained by plotting log of apparent absorbance of light versus logof incident wavelength of the light. The wavelength used is betweenabout 330 nm and 420 nm. A preparation is identified as colloidallystable if a turbidity parameter of less than −3 is determined. Apreparation is identified as colloidally unstable if a turbidityparameter of greater than or equal to −3 is determined.

[0062] The turbidity parameter of the compacted nucleic acid particleswas assessed before and after lyophilization using various excipients,counterions, and with or without polyethylene glycol. See FIG. 12.Sucrose and trehalose were found to be very effective in maintaining theproperties of the pre-lyophilization particles. PEG-acetate similarlywas effective in maintaining these properties.

Example 7

[0063] Particles were observed under the electron microscope before andafter lyophilization. See FIG. 13. Particles made with CK30-PEG10kacetate in the presence of 0.5 M trehalose look similarly rod-likebefore and after lyophilization and rehydration.

Example 8

[0064] Particles were observed before and after lyophilization andrehydration under the electron microscope. The ellipsoidal particles ofcompacted DNA made with CK30 TFA (counterion) in the presence of 0.5Msucrose look identical before and after lyophilization and rehydration.See FIG. 14.

Example 9

[0065] Gene transfer experiments using lyophilized and rehydratedPLASmin™ complexes were performed, comparing them to pre-lyophilizationpreparations. Luciferase enzyme was encoded by the complexes and itsactivity was measured as a means of monitoring gene transfer. Whilesucrose and trehalose were effective in protecting the gene transferactivity to all particles, particles which contained polyethylene glycol(10 kdal) and acetate as a counterion were surprisingly stable tolyophilization, even in the absence of cryoprotectant excipient(disaccharide). See FIG. 15.

Example 10

[0066] Polylysines having an N-terminal cysteine and exactly 30 or 45lysine residues (CK30 or CK45, respectively) were obtained astrifluoroacetate (TFA) salts by solid-phase synthesis. The cysteineresidue was then used to conjugate polyethylene glycol (MW 10,000) toform PEG-ylated polylysines CK30P10K and CK45P10K. The TFA counterionwas exchanged with acetate, bicarbonate, or chloride by gel filtration.DNA was condensed by these polylysines, dialyzed against 0.9% NaCl, andconcentrated to 1 or 4 mg/ml using centrifugal concentrators beforeanalysis. Plasmid DNA having 5921 bp was comprised of kanamycinresistance and luciferase genes, elongation factor-1α promoter and firstintron, CMV enhancer, RU5 translational enhancer from HTLV I, SV40 latepolyadenylation site, and ColE1 origin of replication was used.

[0067] Colloidal stability for the DNA complexes was determined bymeasuring sedimentation of condensed DNA during centrifugation (3,400for 1 min) and scattering of light (turbidity) in the wavelength rangeof 330-415 nm. The turbidity parameter is the slope of a straight lineobtained by plotting log of apparent absorbance (due to scattering) vs.log of incident wavelength in a range outside the true absorption by DNAor peptides (330-415 nm). According to the Rayleigh law, particles thatare small compared to the wavelength of light should have TurbidityParameter of −4. Larger particles, however, scatter light differentlyand have Turbidity Parameters in the range of ˜−1 to −3. Very largeaggregates, have a Turbidity Parameter of ˜−1. We have found that allthe tested DNA formulations were colloidally stable in normal saline(0.9% NaCl) as judged by sedimentation and turbidity measurements. Wealso found that the ability of polylysines to condense DNA depends ontype of associated counterions and length of polylysine. CK30P10k withchloride represents the extreme case since it does not condense DNA orcondenses it very poorly. (FIG. 16).

Example 11

[0068] DNA compacted by CK30P10K with various counterions waselectrophoresed through an agarose gel to examine the effect ofcounterion on net charge of condensed DNA. DNA samples were loadeddirectly on the gel (1.5 μg) or after trypsin treatment for 40 min (0.2μg) to remove polylysine and visualize DNA integrity and relativequantities of supercoiled, nicked, and linear plasmid forms. DNA eithermigrated to the cathode (CK30/acetate, CK30/bicarbonate, CK45/chloride),remained in the well (CK30/TFA), or migrated to the anode(CK30/chloride). (FIG. 18). Therefore, counterions influence effectivenet charge of condensed DNA as visualized by gel electrophoresis.Acetate and bicarbonate bound to CK30P10k and chloride bound to CK45P10kresult in slightly positive net charge, while TFA results inelectrically neutral complexes.

[0069] Serum stability was also evaluated for each of the compacted DNAcomplexes. This was assessed by incubating DNA samples with 75% mouseserum at 37° C. for 2 hr, removing polylysine by trypsinization, andevaluating DNA integrity by gel electrophoresis. Under these conditions,properly condensed DNA is stable, although some nicking andlinearization (very little) occurs. Naked DNA, on the other hand, iscompletely digested within a few minutes (FIG. 18). We found that theability of polylysines to condense and protect DNA depends on type ofassociated counterions and length of polylysine. CK30P10k with chlorideagain represents the extreme case since it does not condense DNA orcondenses it very poorly and does not protect against nucleases.

Example 12

[0070] Intramuscular gene delivery was assessed for each of thecounterion forms of CK30P10K. Fifty μl of DNA was injected intoquadriceps of each leg of CD-1 mice (4-6 weeks old). The total dose was100 μg. Prior to the injection, the animals were anesthetized byintraperitoneal injection of a rodent cocktail of Ketamine, Xylazine,and Acepromazine. One day after the injection, the mice were terminatedand entire quadrceps removed and processed. Protein and luciferaseactivity were determined. (FIG. 19).

[0071] The morphology of the compacted DNA complexes appears to haveinfluenced their in vivo transfection efficiency. CK30/TFA gave thelowest expression (RLU/mg protein), CK30/acetate and CK30/bicarbonate(more relaxed structures) gave 10-100-fold higher RLU/mg, andCK30/chloride gave the expression at the level of naked DNA (same as or10-fold higher than CK30/acetate, depending on harvest day). We havefound that naked DNA is more efficient than condensed DNA and the TFAformulation is much less efficient than other forms of condensed DNA forintramuscular gene delivery.

Example 13

[0072] Intranasal gene delivery was assessed for each of the counterionforms of CK30P10K. Twenty five μl of DNA was administered in 5-μlaliquots into nostrils of C57/BL6 mice using an automated pipette. Thetotal dose was 100 μg. Prior to the injection, the animals wereanesthetized by intraperitoneal injection of a rodent cocktail ofKetamine, Xylazine, and Acepromazine. Two days after the injection, themice were terminated and entire lungs removed and processed. Protein andluciferase activity were determined (FIG. 20). In intranasalapplication, the acetate, bicarbonate, and TFA formulations of condensedDNA are the most efficient among the tested formulations, and naked DNAand CK45/chloride were much less effective. We also found that condensedDNA administered intranasally in water is about 10-fold less efficientthan the same DNA administered in saline.

[0073] Literature Cited

[0074] 1. Cooper, M. J. (1996) Non-infectious gene transfer andexpression systems for cancer gene therapy.

[0075] 2. Semin.Oncol. 23:172-188 Weiss, R. and Nelson, D. WashingtonPost, Sep. 29, 1999, page A1.

[0076] 3. Takeshita, S., Gai, D., Leclerc, G., Pickering, J. G.,Riesssen, R., Wier, L., and Isner, J. M. (1994) Increased geneexpression after liposome-mediated arterial gene transfer associatedwith intimal smooth muscle cell proliferation. J. Clin. Invest.93:652-661.

[0077] 4. Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, D. A.,and Welsh, M. J. (1995) Cellular and molecular barriers to gene transferby a cationic lipid. J. Biol. Chem. 270:18997-19007.

[0078] 5. Wilke, M., Fortunati, E., van den Broek, M., Hoogeveen, A. T.,and Scholte, B. J. (1996) Efficacy of a peptide-based gene deliverysystem depends on mitotic activity. Gene Ther. 3:1133-1142.

[0079] 6. Fasbender, A., Zabner, J., Zeiher, B. G., and Welsh, M. J.(1997) A low rate of cell proliferation and reduce DNA uptake limitcationic lipid-mediated gene transfer to primary cultures of ciliatedhuman airway epithelia. Gene Ther. 41173-1180.

[0080] 7. Sebestyen, M. G., Ludtke, J. J., Bassik, M. C., Zhang, G.,Budker, V., Lukhtanov, E. A., Hagstrom, J. E., and Wolff. J. A. (1998)DNA vector chemistry: the covalent attachment of signal peptides toplasmid DNA. Nat. Biotechnol. 16:80-85.

[0081] 8. Jiang, C., O'Connor, S. P., Fang, S. L., Wang, K. X.,Marshall, J., Williams, J. L., Wilburn, B., Echelard, Y., and Cheng, S.(1998) Efficiency of cationic lipid-mediated transfection of polarizedand differentiated airway epithelial cells in vitro and in vivo.

[0082] 9. Tseng, W. C., Haselton, F. R., and Giorgio, T. D. (1999)Mitosis enhances transgene expression of plasmid delivered by cationicliposomes. Biochim. Biophy. Acta 1445:53-64.

[0083] 10. Mortimer, J., Tam, P., MacLachlan, I., Graham, R. W.,Saravolac, E. G., and Joshi, P. B. (1999) Cationic lipid-mediatedtransfection of cells in culture requires mitotic activity. Gene Ther.6:403-411.

[0084] 11. Mirzayans, R., Aubin, R., and Paterson, M. (1992)Differential expression and stability of foreign genes introduced intohuman fibroblasts by nuclear versus cytoplasmic microinjection. Mutat.Res. 281:115-122.

[0085] 12. Dworetzky, S. I. and Feldherr, C. M. (1988) Translocation ofRNA-coated gold particles through the nuclear pores of oocytes. J. CellBiol. 106:575-584.

[0086] 13. Feldherr, C. M. and Akin D. (1991) Signal-mediated nucleartransport in proliferating and growth-arrested BALB/c 3T3 cells. J. CellBiol. 115:933-939.

1. A method of estimating the colloidal stability of a preparation ofcompacted nucleic acids, comprising the steps of: determining aturbidity parameter of a solution of compacted nucleic acid, wherein theturbidity parameter is defined as the slope of a straight line obtainedby plotting log of apparent absorbance of light versus log of incidentwavelength of the light, wherein said wavelength is between about 330 nmand 420 nm; identifying the preparation as colloidally stable if aturbidity parameter of less than −3 is determined and identifying thepreparation as colloidally unstable if a turbidity parameter of greaterthan or equal to −3 is determined.
 2. A non-naturally occurringcomposition comprising unaggregated nucleic acid complexes, each complexconsisting essentially of a single nucleic acid molecule and one or morepolycation molecules, said polycation molecules having a counterionselected from the group consisting of acetate, bicarbonate, andchloride, wherein said complex is compacted to a diameter which is lessthan (a) double the theoretical diameter of a complex of said singlenucleic acid molecule and a sufficient number of polycation molecules toprovide a charge ratio of about 1:1, in the form of a condensed sphere,or (b) 30 nm, whichever is larger.
 3. The composition of claim 2 whereinthe polycation molecules are polylysine or a polylysine derivative. 4.The composition of claim 3 wherein the polylysine derivative ispolylysine peptide with a cysteine residue.
 5. The composition of claim2, said complex is compacted to a diameter of less than 90 nm.
 6. Thecomposition of claim 2, wherein the nucleic acid complex is compacted toa diameter less than 30 nm.
 7. The composition of claim 2, wherein thenucleic acid complex is compacted to a diameter less than 23 nm.
 8. Thecomposition of claim 2, wherein the nucleic acid complex is compacted toa diameter not more than 12 nm.
 9. The composition of claim 2 whereinsaid complex is compacted to a diameter which is less than double thetheoretical diameter of a complex of said single nucleic acid and asufficient number of positively charged residues to provide a chargeratio of about 1:1, in the form of a condensed sphere.
 10. A method ofpreparing a composition according to claim 2 which comprises mixing thenucleic acid with the polycation having acetate as a counterion, at asalt concentration sufficient for compaction of the complex.
 11. Themethod of claim 10 in which the mixing is monitored to detect, preventor correct, the formation of aggregated or relaxed complexes.
 12. Themethod of claim 10 wherein the salt is NaCl.
 13. The method of claim 10wherein the nucleic acid and the polycation are each, at the time ofmixing, in a solution having a salt concentration of 0.05 to 1.5 M. 14.The method of claim 10 in which the molar ratio of the phosphate groupsof the nucleic acid to the positively charged groups of the polycationis in the range of 4:1 to 1:4.
 15. The method of claim 10 in which thepolycation is added to the nucleic acid, while vortexing at high speed.16. The method of claim 10 in which the nucleic acid is added to thepolycation, while vortexing at high speed.
 17. The method of claim 10wherein the mixing is monitored by a method selected from the groupconsisting of electron microscopy, light scattering, circular dichroism,and absorbance measurement.
 18. The method of claim 10 wherein thepolycation molecules are polylysine or a polylysine derivative.
 19. Themethod of claim 18 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 20. A non-naturally occurringcomposition comprising unaggregated nucleic acid complexes, each complexconsisting essentially of a single nucleic acid molecule and one or morepolycation molecules, wherein said polycation molecules have acounterion selected from the group consisting of acetate, bicarbonate,and chloride, said polycation molecule having a nucleic acid bindingmoiety through which it is complexed to the nucleic acid, wherein saidnucleic acid molecule encodes at least one functional protein, whereinsaid complex is compacted to a diameter which is less than double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere, or 30 nm,whichever is larger.
 21. The composition of claim 20 wherein thepolycation molecules are polylysine or a polylysine derivative.
 22. Thecomposition of claim 21 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 23. The non-naturally occurringcomposition of claim 20 wherein said nucleic acid molecule comprises apromoter which controls transcription of an RNA molecule encoding thefunctional protein.
 24. The non-naturally occurring composition of claim20 wherein the protein is therapeutic.
 25. The non-naturally occurringcomposition of claim 20 wherein the complex is compacted to a diameterwhich is less than 50 nm.
 26. The non-naturally occurring composition ofclaim 20 wherein the complex is compacted to a diameter which is lessthan 30 nm.
 27. The non-naturally occurring composition of claim 20wherein the nucleic acid complex is compacted to a diameter less than 23nm.
 28. The non-naturally occurring composition of claim 20 wherein thenucleic acid complex is compacted to a diameter not more than 12 nm. 29.A non-naturally occurring composition comprising unaggregated nucleicacid complexes, each complex consisting essentially of a singledouble-stranded cDNA molecule and one or more polycation molecules, saidpolycation molecules having a counterion selected from the groupconsisting of acetate, bicarbonate, and chloride, wherein said cDNAmolecule encodes at least one functional protein, wherein said complexis compacted to a diameter which is less than double the theoreticalminimum diameter of a complex of said single cDNA molecule and asufficient number of polycation molecules to provide a charge ratio ofabout 1:1, in the form of a condensed sphere, or 30 nm, whichever islarger.
 30. The composition of claim 29 wherein the polycation moleculesare polylysine or a polylysine derivative.
 31. The composition of claim30 wherein the polylysine derivative is polylysine peptide with acysteine residue.
 32. A non-naturally occurring composition comprisingunaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more polycation molecules,said polycation molecules having a counterion selected from the groupconsisting of acetate, bicarbonate, and chloride, wherein said nucleicacid molecule encodes at least one antisense nucleic acid, wherein saidcomplex is compacted to a diameter which is less than double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere, or 30 nm,whichever is larger.
 33. The composition of claim 32 wherein thepolycation molecules are polylysine or a polylysine derivative.
 34. Thecomposition of claim 33 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 35. A non-naturally occurringcomposition comprising unaggregated nucleic acid complexes, each complexconsisting essentially of a single nucleic acid molecule and one or morepolycation molecules, said polycation molecule having a counterionselected from the group consisting of acetate, bicarbonate, andchloride, wherein said nucleic acid molecule is an RNA molecule, whereinsaid complex is compacted to a diameter which is less than double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere, or 30 nm,whichever is larger.
 36. The composition of claim 35 wherein thepolycation molecules are polylysine or a polylysine derivative.
 37. Thecomposition of claim 36 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 38. A method of preparing a compositioncomprising unaggregated nucleic acid complexes, each complex consistingessentially of a single nucleic acid molecule and one or more polycationmolecules, said method comprising: mixing a nucleic acid molecule with apolycation molecule at a salt concentration sufficient for compaction ofthe complex to a diameter which is less than double the theoreticalminimum diameter of a complex of said single nucleic acid molecule and asufficient number of polycation molecules to provide a charge ratio ofabout 1:1, in the form of a condensed sphere, or 30 nm, whichever islarger, whereby unaggregated nucleic acid complexes are formed, whereineach complex consists essentially of a single nucleic acid molecule andone or more polycation molecules, and wherein said polycation moleculeshave a counterion selected from the group consisting of bicarbonate andchloride.
 39. The method of claim 38 wherein the polycation moleculesare polylysine or a polylysine derivative.
 40. The method of claim 39wherein the polylysine derivative is polylysine peptide with a cysteineresidue.
 41. A method of preparing a composition comprising unaggregatednucleic acid complexes, each complex consisting essentially of a singlenucleic acid molecule and one or more polycation molecules, said methodcomprising: mixing a nucleic acid molecule with a polycation molecule ina solvent to form a complex, said mixing being performed in the absenceof added salt, whereby the nucleic acid forms soluble complexes with thepolycation molecule without forming aggregates, wherein each complexconsists essentially of a single nucleic acid molecule and one or morepolycation molecules, wherein the complexes have a diameter which isless than double the theoretical minimum diameter of a complex of saidsingle nucleic acid molecule and a sufficient number of polycationmolecules to provide a charge ratio of about 1:1, in the form of acondensed sphere, or 30 nm, whichever is larger, wherein the polycationhas acetate as a counterion.
 42. The method of claim 41 wherein thepolycation molecules are polylysine or a polylysine derivative.
 43. Themethod of claim 42 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 44. A method of preparing a compositioncomprising unaggregated nucleic acid complexes, each complex consistingessentially of a single nucleic acid molecule and one or more polycationmolecules, said method comprising: mixing a nucleic acid molecule with apolycation molecule in a solvent to form a complex, said mixing beingperformed in the absence of added salt, whereby the nucleic acid formssoluble complexes with the polycation molecule without formingaggregates, wherein each complex consists essentially of a singlenucleic acid molecule and one or more polycation molecules, wherein thecomplexes have a diameter which is less than double the theoreticalminimum diameter of a complex of said single nucleic acid molecule and asufficient number of polycation molecules to provide a charge ratio ofabout 1:1, in the form of a condensed sphere, or 30 nm, whichever islarger, wherein the polycation has a counterion selected from the groupconsisting of bicarbonate and chloride.
 45. The method of claim 44wherein the polycation molecules are polylysine or a polylysinederivative.
 46. The method of claim 45 wherein the polylysine derivativeis polylysine peptide with a cysteine residue.
 47. Non-naturallyoccurring, soluble compacted complexes of a nucleic acid and apolycation molecule made by the process of claim
 10. 48. Non-naturallyoccurring, soluble compacted complexes of a nucleic acid and apolycation molecule made by the process of claim
 38. 49. Non-naturallyoccurring, soluble compacted complexes of a nucleic acid and apolycation molecule made by the process of claim
 41. 50. Non-naturallyoccurring, soluble compacted complexes of a nucleic acid and apolycation made by the process of claim
 44. 51. The complexes of claim47 wherein the polycation molecules are polylysine or a polylysinederivative.
 52. The complexes of claim 51 wherein the polylysinederivative is polylysine peptide with a cysteine residue
 53. Thecomplexes of claim 48 wherein the polycation molecules are polylysine ora polylysine derivative.
 54. The complexes of claim 53 wherein thepolylysine derivative is polylysine peptide with a cysteine residue. 55.The complexes of claim 49 wherein the polycation molecules arepolylysine or a polylysine derivative.
 56. The complexes of claim 55wherein the polylysine derivative is polylysine peptide with a cysteineresidue.
 57. The complexes of claim 50 wherein the polycation moleculesare polylysine or a polylysine derivative.
 58. The complexes of claim 57wherein the polylysine derivative is polylysine peptide with a cysteineresidue.
 59. A method of preventing or treating a disease or otherclinical condition in a subject which comprises: administeringintramuscularly or to the lung of the subject a prophylactically ortherapeutically effective amount of a composition comprising:unaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more polycation molecules,said polycation molecule having acetate as a counterion, wherein saidcomplex is compacted to a diameter which is less than (a) double thetheoretical minimum diameter of a complex of said single nucleic acidmolecule and a sufficient number of polycation molecules to provide acharge ratio of about 1:1, in the form of a condensed sphere, or (b) 30nm, whichever is larger, said nucleic acid being one whose integration,hybridization or expression within target cells of said subject preventsor treats said disease or other clinical condition.
 60. The method ofclaim 59 wherein the step of administering is by inhalation.
 61. Themethod of claim 59 wherein the step of administering is by intramuscularinjection.
 62. The method of claim 59 wherein the polycation moleculesare polylysine or a polylysine derivative.
 63. The method of claim 62wherein the polylysine derivative is polylysine peptide with a cysteineresidue.
 64. A method of preventing or treating a disease or otherclinical condition in a subject which comprises: administeringintramuscularly or to the lung of the subject a prophylactically ortherapeutically effective amount of a composition comprising:unaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more polycation molecules,said polycation molecule having a counterion selected from the groupconsisting of bicarbonate and chloride, wherein said complex iscompacted to a diameter which is less than (a) double the theoreticalminimum diameter of a complex of said single nucleic acid molecule and asufficient number of polycation molecules to provide a charge ratio ofabout 1:1, in the form of a condensed sphere, or (b) 30 nm, whichever islarger, said nucleic acid being one whose integration, hybridization orexpression within target cells of said subject prevents or treats saiddisease or other clinical condition.
 65. The method of claim 64 whereinthe polycation molecules are polylysine or a polylysine derivative. 66.The method of claim 65 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 67. The method of claim 64 wherein thestep of administering is by inhalation.
 68. The method of claim 64wherein the step of administering is by intramuscular injection.
 69. Thecomposition of claim 20 wherein said complex is compacted to a diameterwhich is less than double the theoretical diameter of a complex of saidsingle nucleic acid and a sufficient number of positively chargedresidues to provide a charge ratio of about 1:1, in the form of acondensed sphere.
 70. The composition of claim 29 wherein the nucleicacid complexes are associated with a lipid.
 71. The composition of claim29 wherein said complex is compacted to a diameter of less than 90 nm.72. The composition of claim 29 wherein the nucleic acid complex iscompacted to a diameter less than 30 nm.
 73. The composition of claim 29wherein the nucleic acid complex is compacted to a diameter less than 23nm.
 74. The composition of claim 29 wherein the nucleic acid complex iscompacted to a diameter not more than 12 nm.
 75. The composition ofclaim 29 wherein said complex is compacted to a diameter which is lessthan double the theoretical diameter of a complex of said single nucleicacid and a sufficient number of positively charged residues to provide acharge ratio of about 1:1, in the form of a condensed sphere.
 76. Thecomposition of claim 32 wherein said complex is compacted to a diameterof less than 90 nm.
 77. The composition of claim 32 wherein the nucleicacid complex is compacted to a diameter less than 30 nm.
 78. Thecomposition of claim 32 wherein the nucleic acid complex is compacted toa diameter less than 23 nm.
 79. The composition of claim 32 wherein thenucleic acid complex is compacted to a diameter not more than 12 nm. 80.The composition of claim 32 wherein said complex is compacted to adiameter which is less than double the theoretical diameter of a complexof said single nucleic acid and a sufficient number of positivelycharged residues to provide a charge ratio of about 1:1, in the form ofa condensed sphere.
 81. The composition of claim 35 said complex iscompacted to a diameter of less than 90 nm.
 82. The composition of claim35 wherein the nucleic acid complex is compacted to a diameter less than30 nm.
 83. The composition of claim 35 wherein the nucleic acid complexis compacted to a diameter less than 23 nm.
 84. The composition of claim35 wherein the nucleic acid complex is compacted to a diameter not morethan 12 nm.
 85. The composition of claim 35 wherein said complex iscompacted to a diameter which is less than double the theoreticaldiameter of a complex of said single nucleic acid and a sufficientnumber of positively charged residues to provide a charge ratio of about1:1, in the form of a condensed sphere.
 86. The method of claim 38wherein the salt is NaCl.
 87. The method of claim 38 wherein the nucleicacid and the polycation are each, at the time of mixing, in a solutionhaving a salt concentration of 0.05 to 1.5 M.
 88. The method of claim 38in which the mixing is monitored to detect, prevent or correct, theformation of aggregated or relaxed complexes.
 89. The method of claim 38in which the molar ratio of the phosphate groups of the nucleic acid tothe positively charged groups of the polycation is in the range of 4:1to 1:4.
 90. The method of claim 38 in which the polycation is added tothe nucleic acid, while vortexing at high speed.
 91. The method of claim38 in which the nucleic acid is added to the polycation, while vortexingat high speed.
 92. The method of claim 38 wherein the mixing ismonitored by a method selected from the group consisting of electronmicroscopy, light scattering, circular dichroism, and absorbancemeasurement.
 93. The method of claim 41 in which the mixing is monitoredto detect, prevent or correct, the formation of aggregated or relaxedcomplexes.
 94. The method of claim 41 in which the molar ratio of thephosphate groups of the nucleic acid to the positively charged groups ofthe polycation is in the range of 4:1 to 1:4.
 95. The method of claim 41in which the polycation is added to the nucleic acid, while vortexing athigh speed.
 96. The method of claim 41 in which the nucleic acid isadded to the polycation, while vortexing at high speed.
 97. The methodof claim 41 wherein the mixing is monitored by a method selected fromthe group consisting of electron microscopy, light scattering, circulardichroism, and absorbance measurement.
 98. The method of claim 44 inwhich the mixing is monitored to detect, prevent or correct, theformation of aggregated or relaxed complexes.
 99. The method of claim 44in which the molar ratio of the phosphate groups of the nucleic acid tothe positively charged groups of the polycation is in the range of 4:1to 1:4.
 100. The method of claim 44 in which the polycation is added tothe nucleic acid, while vortexing at high speed.
 101. The method ofclaim 44 in which the nucleic acid is added to the polycation, whilevortexing at high speed.
 102. The method of claim 44 wherein the mixingis monitored by a method selected from the group consisting of electronmicroscopy, light scattering, circular dichroism, and absorbancemeasurement.
 103. A non-naturally occurring composition comprisingunaggregated nucleic acid complexes, each complex consisting essentiallyof a single nucleic acid molecule and one or more polycation molecules,said polycation molecules having a counterion selected from the groupconsisting of acetate, bicarbonate, and chloride.
 104. The compositionof claim 103 wherein the counterion is acetate.
 105. The composition ofclaim 2 wherein said polycation is CK15-60P10 and the counterion isacetate, wherein CK15-60P10 is a polyamino acid polymer of oneN-terminal cysteine and 15-60 lysine residues, wherein a molecule ofpolyethylene glycol having an average molecular weight of 10 kdal isattached to the cysteine residue.
 106. The composition of claim 105wherein the polycation molecule comprises 30 residues of lysine. 107.The composition of claim 105 wherein the polycation molecule comprises atargeting moiety.
 108. The composition of claim 105, said complex iscompacted to a diameter of less than 90 nm.
 109. The composition ofclaim 105, wherein the nucleic acid complex is compacted to a diameterless than 30 nm.
 110. The composition of claim 105, wherein the nucleicacid complex is compacted to a diameter less than 23 nm.
 111. Thecomposition of claim 105, wherein the nucleic acid complex is compactedto a diameter not more than 12 nm.
 112. The composition of claim 105wherein said complex is compacted to a diameter which is less thandouble the theoretical diameter of a complex of said single nucleic acidand a sufficient number of positively charged residues to provide acharge ratio of about 1:1, in the form of a condensed sphere.
 113. Thecomposition of claim 105 which is lyophilized.
 114. The composition ofclaim 105 which is rehydrated after lyophilization.
 115. The compositionof claim 105 which does not contain a disaccharide.
 116. A method ofdelivering polynucleotides to cells comprising: contacting thecomposition of claim 114 with cells, whereby the nucleic acid isdelivered to and taken up by the cells.
 117. The method of claim 116wherein the composition does not contain a disaccharide.
 118. Thecomposition of claim 20 wherein the polycation is CK15-60P10, and thecounterion is acetate, wherein CK15-60 is a polyamino acid polymer ofone N-terminal cysteine and 15-60 lysine residues, wherein a molecule ofpolyethylene glycol having an average molecular weight of 10 kdal isattached to the cysteine residue.
 119. The composition of claim 118wherein the polycation molecule comprises 30 residues of lysine. 120.The composition of claim 118 wherein the polycation molecule comprises atargeting moiety.
 121. The composition of claim 118 which islyophilized.
 122. The non-naturally occurring composition of claim 118wherein said nucleic acid molecule comprises a promoter which controlstranscription of an RNA molecule encoding the functional protein. 123.The non-naturally occurring composition of claim 118 wherein the proteinis therapeutic.
 124. The non-naturally occurring composition of claim118 wherein the complex is compacted to a diameter which is less than 50nm.
 125. The non-naturally occurring composition of claim 118 whereinthe complex is compacted to a diameter which is less than 30 nm. 126.The non-naturally occurring composition of claim 118 wherein the nucleicacid complex is compacted to a diameter less than 23 nm.
 127. Thenon-naturally occurring composition of claim 118 wherein the nucleicacid complex is compacted to a diameter not more than 12 nm.
 128. Thecomposition of claim 118 wherein said complex is compacted to a diameterwhich is less than double the theoretical diameter of a complex of saidsingle nucleic acid and a sufficient number of positively chargedresidues to provide a charge ratio of about 1:1, in the form of acondensed sphere.
 129. The composition of claim 118 which is rehydratedafter lyophilization.
 130. The composition of claim 118 which does notcontain a disaccharide.
 131. A method of delivering polynucleotides tocells comprising: contacting the composition of claim 129 with cells,wherein the polynucleotide encodes a protein, whereby the protein isexpressed.
 132. The composition of claim 29 wherein said polycation isCK15-60P10, and said counterion is acetate, wherein CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, wherein a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal is attached to the cysteine residue. 133.The composition of claim 132 wherein the polycation molecule comprises30 residues of lysine.
 134. The composition of claim 132 wherein thepolycation molecule comprises a targeting moiety.
 135. The compositionof claim 132 which is lyophilized.
 136. The composition of claim 132wherein said complex is compacted to a diameter which is less thandouble the theoretical diameter of a complex of said single nucleic acidand a sufficient number of positively charged residues to provide acharge ratio of about 1:1, in the form of a condensed sphere.
 137. Thecomposition of claim 132 which is rehydrated after lyophilization. 138.The composition of claim 132 which does not contain a disaccharide. 139.A method of delivering polynucleotides to cells comprising: contactingthe composition of claim 137 with cells, wherein the polynucleotideencodes a protein, whereby the protein is expressed.
 140. Thecomposition of claim 32 wherein said polycation is CK15-60P10, and thecounterion is acetate, wherein CK15-60P10 is a polyamino acid polymer ofone N-terminal cysteine and 15-60 lysine residues, wherein a molecule ofpolyethylene glycol having an average molecular weight of 10 kdal isattached to the cysteine residue.
 141. The composition of claim 140wherein the polycation molecule comprises 30 residues of lysine. 142.The composition of claim 140 wherein the polycation molecule comprises atargeting moiety.
 143. The composition of claim 140 which islyophilized.
 144. The composition of claim 140 wherein said complex iscompacted to a diameter which is less than double the theoreticaldiameter of a complex of said single nucleic acid and a sufficientnumber of positively charged residues to provide a charge ratio of about1:1, in the form of a condensed sphere.
 145. The composition of claim140 which is rehydrated after lyophilization.
 146. The composition ofclaim 140 which does not contain a disaccharide.
 147. A method ofdelivering polynucleotides to cells comprising: contacting thecompositions of claim 145 with cells, wherein the polynucleotide encodesan antisense nucleic acid, whereby the antisense nucleic acid isexpressed.
 148. The composition of claim 35 wherein said polycation isCK15-60P10, and said counterion is acetate, wherein CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, wherein a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal is attached to the cysteine residue. 149.The composition of claim 148 wherein the polycation molecule comprises30 residues of lysine.
 150. The composition of claim 148 wherein thepolycation molecule comprises a targeting moiety.
 151. The compositionof claim 148 which is lyophilized.
 152. The composition of claim 148which is lyophilized and rehydrated.
 153. The composition of claim 148which does not contain a disaccharide.
 154. A method of deliveringpolynucleotides to cells comprising: contacting the composition of claim152 with cells, whereby the polynucleotide is delivered to and taken upby the cells.
 155. The method of claim 41, wherein said polycation isCK15-60P10, and said counterion is acetate, wherein CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, wherein a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal is attached to the cysteine residue. 156.The method of claim 155 further comprising lyophilizing the unaggregatednucleic acid complexes.
 157. The method of claim 156 further comprisingrehydrating the lyophilized nucleic acid complexes.
 158. The method ofclaim 155 wherein the polycation molecule comprises 30 residues oflysine.
 159. The method of claim 155 wherein the polycation moleculecomprises a targeting moiety.
 160. A method of preparing a compositioncomprising unaggregated nucleic acid complexes, each complex consistingessentially of a single nucleic acid molecule and one or more polycationmolecules, said method comprising: mixing a nucleic acid molecule with apolycation molecule at a salt concentration sufficient for compaction ofthe complex to a diameter which is less than double the theoreticalminimum diameter of a complex of said single nucleic acid molecule and asufficient number of polycation molecules to provide a charge ratio ofabout 1:1, in the form of a condensed sphere, or 30 nm, whichever islarger, whereby unaggregated nucleic acid complexes are formed, whereineach complex consists essentially of a single nucleic acid molecule andone or more polycation molecules, and wherein said polycation moleculeshave a counterion selected from the group consisting of acetate,bicarbonate and chloride.
 161. The method of claim 160 wherein thecounterion is acetate.
 162. The method of claim 160 wherein thepolycation molecules are polylysine or a polylysine derivative.
 163. Themethod of claim 162 wherein the polylysine derivative is polylysinepeptide with a cysteine residue.
 164. Non-naturally occurring, solublecompacted complexes of a nucleic acid and a polycation molecule made bythe method of claim
 160. 165. The method of claim 160 wherein the saltis NaCl.
 166. The method of claim 160 wherein the nucleic acid and thepolycation are each, at the time of mixing, in a solution having a saltconcentration of 0.05 to 1.5 M.
 167. The method of claim 160 in whichthe mixing is monitored to detect, prevent or correct, the formation ofaggregated or relaxed complexes.
 168. The method of claim 160 in whichthe molar ratio of the phosphate groups of the nucleic acid to thepositively charged groups of the polycation is in the range of 4:1 to1:4.
 169. The method of claim 160 in which the polycation is added tothe nucleic acid, while vortexing at high speed.
 170. The method ofclaim 160 in which the nucleic acid is added to the polycation, whilevortexing at high speed.
 171. The method of claim 160 wherein the mixingis monitored by a method selected from the group consisting of electronmicroscopy, light scattering, circular diochroism, and absorbancemeasurement.
 172. The method of claim 160, wherein said polycation isCK15-60P10 and the counterion is acetate, wherein CK15-60P10 is apolyamino acid polymer of one N-terminal cysteine and 15-60 lysineresidues, wherein a molecule of polyethylene glycol having an averagemolecular weight of 10 kdal is attached to the cysteine residue. 173.The method of claim 172 further comprising lyophilizing the unaggregatednucleic acid complexes.
 174. The method of claim 173 further comprisingrehydrating the lyophilized nucleic acid complexes.
 175. The method ofclaim 172 wherein the polycation molecule comprises 30 residues oflysine.
 176. The method of claim 172 wherein the polycation moleculecomprises a targeting moiety.